Impaired insulin signaling in the myocardium may contribute to cardiac dysfunction in obesity, type 2 diabetes and other insulin resistant states. In the previous funding cycle of this award we demonstrated that myocardial insulin and IGF-1 signaling regulates mitochondrial function in the heart, and that insulin signaling is required for maintaining myocardial viability in the face of hemodynamic stressors such as ischemia and pathological cardiac hypertrophy. Moreover, we identified a central role for PI3K signaling in coordinating the mitochondrial adaptation to physiological cardiac hypertrophy. To further dissect molecular pathways that govern insulin and growth factor signaling in the heart we generated mice with cardiomyocyte-restricted deletion of the insulin receptor substrates IRS-1 and IRS-2 (CIRS12KO). These animals develop heart failure shortly after weaning, on the basis of unregulated autophagy, thereby defining an essential role for insulin and IGF-1 signaling acting via IRS proteins in the regulation cardiac autophagy in vivo. Although an increase in cardiac autophagy is essential for survival in the perinatal period prior to establishing regular feeding, it is effectively repressed once feeding ensues. Our observations strongly implicate insulin as the signal that mediates this process in the heart. Thus in the absence of IRS1and IRS2 the ability of insulin to repress autophagy is abrogated and unrestrained autophagy occurs with devastating consequences on the neonatal heart. Our preliminary studies also indicate that in adult hearts that lack insulin receptors and hearts from STZ-diabetic mice exhibit increased autophagy. In the next phase of these studies we will perform experiments to elucidate the molecular mechanisms by which insulin and IGF-1 signaling regulates autophagy in the heart, and elucidate the role of autophagy in the pathogenesis of diabetic cardiomyopathy. Aim 1: will delineate the proximal and distal signaling modules that are required for the regulation of cardiomyocyte autophagy by insulin signaling in vivo. We hypothesize that insulin suppresses autophagy by activating insulin and IGF-1 receptors, which act via PI3K-PDK1 and Akt1 and 2 to activate mTOR dependent and independent pathways that suppress autophagy. This hypothesis will be tested using mice with (a) combined deletion of IGF-1 and Insulin Receptors in cardiomyocytes, (b) deletion of PDK1 expression in cardiomyocytes, (c) combined deletion of Akt1 and Akt2 in cardiomyocytes and (d) inducible expression of Akt1. Aim 2: will determine the contribution of increased autophagic flux to adverse cardiac remodeling following pressure overload or ischemic injury in genetically insulin resistant hearts or in hearts from mice with type 1 or type 2 diabetes. We hypothesize that increased autophagy will contribute to adverse left ventricular (LV) remodeling in adult mice with cardiomyocyte-restricted loss of insulin receptors (CIRKO) or in mice with type 1-diabetes in which basal rates of autophagy are increased. Aim 3: will determine the mechanisms by which insulin signaling suppresses cardiomyocyte autophagy in cultured ventricular myocytes or H9C2 myoblasts. These studies will determine the relative contribution of mTOR activation versus phosphorylation of FOXO1 and FOXO3 in suppressing cardiomyocyte autophagy. They will also determine if the suppression of cardiomyocyte autophagy by insulin signaling requires increased uptake of glucose or amino acids. Aim 4: will determine if insulin signaling suppresses cardiomyocyte autophagy by disrupting initiation of autophagic vesicle formation or autophagic vesicle enucleation. Specifically we will determine if insulin signaling mediates an mTOR-dependent inactivation of the mammalian homologs of Atg1 (ULK1 and ULK2) and determine if insulin mediated activation of Akt will increase the association of Beclin1 with Bcl2, thereby inhibiting the activity of the Vps34 complex that promotes autophagic vesicle enucleation. Successful completion of these studies will provide novel insights into the regulation of myocardial autophagy by insulin receptor-mediated signaling. The studies will provide insight into the potential role of altered myocardial autophagy in insulin resistance or diabetes - associated cardiac dysfunction. Moreover, increased knowledge of the regulation of autophagic pathways by insulin or IGF-1 in the heart may lead to novel strategies to modulate myocardial autophagy, which is implicated in the pathophysiology of heart failure following ischemia or pressure overload. PUBLIC HEALTH RELEVANCE: Heart disease remains the leading cause of death in individuals with diabetes. Moreover, the risk of developing heart failure is significantly increased in diabetic individuals even after correcting for underlying atherosclerosis. Multiple mechanisms such as lipotoxicity, glucotoxicity, oxidative stress, impaired myocardial calcium homeostasis and impaired insulin signaling have been implicated. The focus of the work supported by this grant has been to elucidate the potential role that changes in myocardial insulin signal transduction would play in contributing to cardiac dysfunction in obesity and diabetes. Our studies discovered important roles for myocardial insulin signaling in the regulation of myocardial mitochondrial energy metabolism, and a role for insulin signaling in regulating myocardial size and in limiting injury in the presence of hemodynamic stress. We have now obtained evidence for an important role for insulin signaling in the regulation myocardial autophagy. Autophagy is a conserved process of bulk degradation of cytosol and organelles, which at low levels plays an important role in recycling damaged organelles and recycling nutrients particularly during times of nutrient depletion. However, excessive autophagy could contribute to tissue dysfunction. In the continuation of these studies we will determine the mechanisms by which insulin signaling regulates autophagy in the heart and the role of autophagy in diabetic cardiomyopathy. Knowledge gained from these studies will set the stage for determining the role that altered autophagy may play in the pathophysiology of cardiac dysfunction in obesity, insulin resistance or diabetes. Moreover, elucidation of the signaling mechanisms that regulate autophagy in cardiomyocytes may lead to novel therapies for preventing or modulating heart injury as occurs in diabetes, cardiac ischemia or cardiac hypertrophy.